Naturally occurring antibodies and monoclonal antibodies have two antigen binding sites which recognize the same antigen. In contrast, bifunctional antibodies, also referred to as heterobispecific antibodies, are synthetically or genetically engineered molecules that are capable of binding to two antigenic determinants. The ability to bind to the two different antigens resides in one molecule.
Bifunctional antibodies were first produced by fusing two different monoclonal antibody-producing hybridomas, which each recognized a different antigenic sites. According to this method each hybridoma is characterized by a different selectable marker, sensitivity to hypoxanthine-aminopterin-thymidine (HAT medium) and resistance to azaguanine [Milstein and Cuello, Nature, 305:537-540 (1983)]. The fused hybridomas are capable of synthesizing two different heavy chains and two different light chains, so that theoretically there are ten different combinations which can be formed to produce an antibody containing two heavy chains associated with two light chains.
However, only one of these antibodies will be bifunctional and must be purified from the other forms. The bifunctional antibody will form an even smaller proportion of the total antibodies if the heavy chains are of different isotypes. A disadvantage of this method is that fused hybridomas are less stable cytogenically than the parent hybridomas and non-fused cells.
These first bifunctional antibodies were used as an alternative to indirect immunocytochemistry, since they avoid the need for direct conjugation of an indicator molecule to the antibody by chemical modification which results in a partial loss of activity and increased danger of nonspecific binding.
Another method for producing bifunctional antibodies has been described using heterobifunctional crosslinkers to chemically link two different monoclonal antibodies, so the aggregate will bind to two different targets [Staerz et al, Nature: 314:628-631 (1985); Perez et al, Nature: 316:354-356 (1985)]. This type of bifunctional antibody has been produced to focus a T-cell response to a chosen target such as a tumor cell or a virally infected cell [Clark and Waldmann, JNCI, 79:1393-1401 (1987); Gilliland et al, Proc. Natl. Acad. Sci. USA, 85:7719-7723 (1988); Staerz et al, Eur. J. Immunol., 17:571-574 (1987)]. A disadvantage of this type of bifunctional antibody is that the chemical heteroconjugates diffuse slowly into tissues and are rapidly removed from the circulation.
Bifunctional antibodies have also been produced by gene transfer into a hybridoma by retrovirus-derived shuttle vectors or selectable plasmids containing light and heavy chain genes [DeMonte et al, Proc. Natl. Acad. Sci., USA, 87:2941-2945 (1990); Lenz and Weidle, Gene: 87:213-218 (1990)]. This method produces a mixture of antibodies from which the bifunctional antibody must be purified. However, these transfected hybridomas are more likely to be stable than fused hybridomas.
Bifunctional antibodies can also be produced by reduction of monoclonal antibodies to the single heavy chain associated with its single light chain (HL form), mixing with a second monoclonal antibody followed by reoxidation to produce mixed antibodies [Staerz and Bevan, Proc. Natl. Acad. Sci., USA, 83:1453-1457 (1986)].
Bifunctional antibodies produced as described above have been employed in a variety of ways. For example, a bifunctional antibody with binding sites for a tumor cell surface antigen and for a T-cell surface receptor, T3 or Ti, will direct the lysis of specific tumor cells by T cells [Clark and Waldman, JNCI, cited above]. A bifunctional antibody with specificity for fibrin and a plasminogen activator has been proposed as capable of increasing the effective concentration of the plasminogen activator in the proximity of a fibrin deposit [Haber et al, Science, 243:51-56 (1989)]. More recently, bifunctional antibodies Which are specific for gliomas and the CD3 epitope on T cells have been successfully used in treating brain tumors in human patients [T. Nitta et al, Lancet, 355:368-371 (1990)].
There remains a need in the art for a method of making recombinant antibodies, particularly bifunctional antibodies which does not require extensive purification steps or chemically controlled conditions.